1. Technical Field
The present invention relates to an apparatus for measuring a fluorescence lifetime, and more specifically to an apparatus for precisely measuring a fluorescence lifetime that is an inherent optical characteristic of fluorescence materials in a short measurement time. This invention provides a means to acquire the fluorescence lifetime information for high-speed measurement applications such as a fluorescence lifetime imaging microscope that acquires an image of spatial distribution of the fluorescence lifetime for a fluorescent sample.
2. Related Art
A molecule can absorb an incident photon as a result of electronic transition from the ground state to a higher energy level when the molecule has an electronic state with the potential energy that matches to the energy of the incident photon. A fluorescence emission phenomenon is observed for a certain kind of molecules, which emit fluorescence photons by the excited electron returning back to the ground state. For fluorescent molecules, each molecule has a characteristic absorption wavelength and fluorescence emission wavelength. Meanwhile, the difference between those two wavelengths is called Stoke's shift and is typically on the order of tens of nanometers in wavelength. In other words, a fluorescence molecule absorbs a photon of excitation light and emits a photon of a slightly longer wavelength or a little less energy than that of the excitation photon. The fluorescence materials can be characterized and specified by the absorption wavelength and the emission wavelength of light. The fluorescence microscopy takes advantage of this specific characteristic of fluorescence materials by using optical filters that distinguish the fluorescence signals from the unwanted noisy excitation background.
The fluorescence microscope equips a light source that irradiates excitation light onto the microscope sample. The wavelength of the excitation light should match to the absorption wavelength of target fluorescent materials that reside in the microscope sample. By using an optical filter, the microscope selectively detects the intensity of the fluorescence signal at a wavelength longer than that of the excitation light.
Two kinds of photo-detectors can be utilized for the purpose: image sensors and single photo-detectors. Image sensors include films and solid-state array photo-detectors such as a charge-coupled device (CCD) and acquire the spatially distributed information of an image by a parallel manner at once. This methodology of image acquisition in microscopy is often called wide-field microscopy and is used for most of the existing conventional microscopes.
On the other hand, the image information is acquired by a scanning methodology. In scanning microscopy such as a confocal microscope, the signals of an image are acquired sequentially by a scan of the measurement point. Single photo-detectors such as a photo-multiplier tube (hereinafter, referred to as PMT) are used for the scanning microscopes.
The fluorescence microscope can be applied to obtain images by taking advantage of an intrinsic fluorescence property of a sample (auto-fluorescence). But it is mainly used to acquire an image of extrinsic fluorescence distribution in biological microscopy applications. Fluorescence dye molecules or fluorescent proteins are diffused and chemically bound to target molecules inside samples such as a cell or tissue. This process of sample preparation is often called labeling or staining. The labeled biological sample is highlighted at the parts stained with the extrinsic fluorescence molecules for a fluorescence microscope to acquire the distribution of a certain kind of the target molecules in the sample.
Most of the conventional fluorescence microscopes acquire image information based on intensity of fluorescence light. But advanced methods of forming images by collecting spectroscopic information other than the intensity of fluorescence are being recently developed. In particular, the information on the fluorescence lifetime is important in the advanced microscopy because it can provide more detailed information on the environments by which the fluorescence materials are surrounded.
Electrons in fluorescence molecules are excited by excitation light and then stay in the excited state for a while. Thereafter, the electrons in the excited state are transited to the ground state emitting a fluorescence photon. The temporal delay between the excitation and emission events for a photon is basically random but obeys a characteristic probability function given by the principle of quantum mechanics.
Before the excited electron being in the fluorescent state, the electron may stay in an intermediate state for a moment. But the excited electron usually relaxes to the fluorescent state almost immediately after being excited with only a few picoseconds for most of fluorescence molecules. This intermediate process is called relaxation of the excited electron. Neglecting this fast relaxation process, the photon emission probability is solely determined by the quantum-mechanical properties of the fluorescence state and the ground state. And each molecule has its own transition characteristic that determines the average time for which the electron stays in the fluorescent state, which is called the fluorescence lifetime.
The transition probability of electrons, that is, the generation probability of the fluorescence photons reaches a peak at the time point of the excitation, neglecting the ultra-fast relaxation process. Thereafter, the transition probability is characterized by an exponential decay curve. The time constant of the exponential decay curve is referred to as the fluorescence lifetime and can be measured by investigating the generation time of a plurality of fluorescence photons.
The fluorescence lifetime is regarded as a characteristic constant of each fluorescence molecule in the case of being isolated. However, the fluorescence lifetime can change according to the environment by which the fluorescence materials are surrounded when the environment provides an effective intermediate state through which the electron can pass. Various ions can provide electrons with such a non-fluorescent pathway to the ground state. Presence of such a competing pathway can decrease the fluorescence emission efficiency and the fluorescence lifetime as compared to the case of the absence.
Fluorescence molecules can play roles of molecular sensors of various ions such as oxygen, hydrogen, calcium or sodium ions, when decreases in the fluorescence efficiency or lifetime can be effectively detected. It is the principle of the fluorescence lifetime imaging microscopy (hereinafter, referred to as FLIM), which investigates the spatial concentration distribution of the above-mentioned ions using the information on the fluorescence lifetime in space. The same principle can be applied to a new class of microscopy technique: Föster resonance energy transfer (hereinafter, referred to as FRET) microscopy. In FRET microscopy, a fluorescence molecule of another kind is used to provide the excited electron with a new fluorescent pathway competing with its own internal pathway. The nanometric distance between the two molecules can be measured by detecting the change of the fluorescence lifetime.
Various image acquisition techniques of FLIM and FRET for wide-field microscopy have been developed by utilizing gated image intensifiers. The gated image intensifiers are the high-speed switchable version of image intensifiers that have fast gating or switching capabilities. They provide a way of measuring a short fluorescence lifetime by taking a series of images with varying temporal delays with respect to the moment of excitation. Because of the parallel signal acquisitions, this wide-field microscopy methodology of fluorescence lifetime measurement can make acquisition of a lifetime image completed within less than a few seconds. However, the scheme of fluorescence lifetime measurement for the wide-field microscopy can not be applied to high-resolution three-dimensional scanning microscopy such as a confocal microscopy due to the nature of parallel signal acquisitions.
On the other hand, the scanning microscopy including the confocal microscope and the multi-photon excitation fluorescence microscope provides better resolutions with depth-sectioning capabilities. This kind of microscopes performs a measurement on a spatial point at a time and scans the measurement point spatially, thereby sequentially obtaining the image information. Signal acquisition of the confocal microscope is performed selectively for a focus of an objective lens of the microscope. The image is constructed by the relative movement of the focus. The confocal microscope utilizes a spatial filter, such as a pinhole, that is placed at the confocal point after the objective lens and distinguishes the fluorescence signal of the objective focal region from the out-of-focus signals. The multi-photon excitation fluorescence microscope can naturally obtain the same effect without using a pinhole because the multi-photon absorption-excitation phenomenon occurs effectively at the focus having a high light intensity as a nonlinear process.
By utilizing a fluorescence lifetime measurement instrument, a scanning microscope can obtain FLIM images. There is little difference between an apparatus for measuring the fluorescence lifetime in the scanning FLIM microscopy and an apparatus for measuring the fluorescence lifetime in the time-resolved spectroscopy. The measurement of a fluorescence lifetime in the typical time-resolved spectroscopy mainly uses a time-correlated single photon counter (Hereinafter, referred to TCSPC) or a phase fluorometer. Those lifetime measurement instruments can be employed by a scanning microscope as a special photo-detector of optical signal sensing to implement a scanning FLIM microscope system.
The measurement of a fluorescence lifetime can be achieved by measuring a plurality of photons generated by a plurality of fluorescence molecules or a plurality of photons generated by exciting a fluorescence molecule many times as in the single-molecule spectroscopy. This is basically a process of analyzing a time-domain intensity of fluorescence that appears to be an exponential decay function. If an infinite number of fluorescence photons are collected and detected, the obtained time-domain fluorescence intensity will equal to the probability distribution function of fluorescence photon emission of the molecules in the case of impulse excitation. When a molecule is excited by a very short excitation pulse as an impulse excitation at t=0, the intensity of fluorescence light or the density of fluorescence photons IF(t) has the following distribution in time.IF(t)=I0e−t/τu(t)where I0 represents an initial value of the function, τ represents the fluorescence lifetime, and u(t) represents the step function of u(t)=0 when t<0 and u(t)=1 when t≧0. In other words, the fluorescence lifetime is the time required for the emission probability of the fluorescence photon to be reduced by a factor of 1/e after the moment of excitation. For organic fluorescence molecules used for biological or medical imaging applications, the lifetimes are typically between 0.1 ns and 5 ns.
The TCSPS detects a single-photon response owing to the high-gain photodetectors like a PMT or an avalanche photo diode (APD). As a photon counter, the TCSPC detects only a single photon at a time and counts the number of detected photons in measuring the detected time or arrival time of the photons. The arrival time of a single photon can precisely be measured by using a constant-fraction discriminator that detects the rising edge of the photo-electronic pulse of the single photon in time. The temporal precision of arrival time determination is even finer than the electronic response time of the photo-detector characterized by the full duration of the impulse response. Thus the TCSPC can provide better temporal resolutions so that it can accurately measure short lifetimes of a few hundreds of picoseconds.
A temporal histogram of fluorescence photon emission detection events is obtained by using the TCSPC photodetection instrument. The histogram shows the characteristic exponential decay function of fluorescence emission with a fine time resolution. In order to obtain a histogram of a good signal-to-noise ratio, more than thousands of photon counts should be detected by the TCSPC. Thereafter, the histogram is considered as the probability distribution function (PDF) of fluorescence emission. The fluorescence lifetime can be extracted from the histogram by various signal processing methods. The most popular method of signal analysis is the curve fitting method, in which the histogram is fitted by an exponential decay function that best matches to the histogram. An alternative analysis method is the mean delay method, in which the expected value of arrival time, that is, the time average of the measured histogram function is calculated. Because the mean temporal delay of an exponential decay function is equal to the lifetime, the fluorescence lifetime is calculated by finding the mean delay with respect to the initial time point of the decay.
Because of superior characteristics of the TCSPC in precision and accuracy of lifetime determination, the TCSPC is widely used in the time-resolved spectroscopy and the scanning FLIM microscopy. However, the TSCPC has a problem of a long measurement time, which is a fundamental problem in the single photon counting method. Since the TCSPC can count only a single photon for each measurement period, the intensity of fluorescence signal should be intentionally reduced so that the number of photons is less than one for each pulse. If more than two photons are sensed by the counter within the measurement period, in particular, if two photons are almost simultaneously arrived so that they cannot be divided into two distinct pulses, the counter detects only the value of the first arrived photon. As a result, the measured fluorescence lifetime is shorter than the actual value due to the signal loss.
Because of the “single photon condition”, the measurement of a fluorescence lifetime in the TCSPC is completed only after the photon counting is performed many times by inputting a plurality of excitation light pulse. Also, the time interval between the excitation light pulses should be sufficiently longer than the fluorescence lifetime to be measured. If the time interval between the excitation light pulses is not sufficiently longer than the fluorescence lifetime, the decay waveforms of two adjacent fluorescence emissions may overlap, making it impossible to obtain an accurate value of the fluorescence lifetime. The pulse period of the excitation light should be, at least, five times longer than the fluorescence lifetime τ. Therefore, if a fluorescence lifetime to be measured is 5 ns, the frequency of the photon count will be lower than 40 MHz (40×106 photons/sec) under the conditions that the excitation light pulse period is longer than 25 ns and one fluorescence photon is counted for each pulse period. Because the emission and measurement probability of a fluorescence photon has a random characteristic, the average number of photons detected for each period should be even smaller than one to ensure the single photon condition. The average number of photons per pulse period should be, at least, 1/10 to make the probability of multiple photons being detected less than 1/100 for an accurate analysis of a fluorescence lifetime. Thus the average frequency of photon counting should be lower than 4 MHz in this condition. And the number of counts required for a lifetime determination is larger than 100 to obtain a random error of less than 10% (<0.5 ns for a lifetime of 5 ns) in standard deviation. Therefore, the measurement rate can not exceed 40 kHz and the measurement period should be larger than 25 μs.
This property of a low measurement speed of the TCSPC limits the image acquisition speed of the scanning FLIM microscope that uses the TCSPC for fluorescence lifetime determination. The microscope image is usually composed of one million pixels. Since it takes longer than 25 μs to measure a fluorescence lifetime for a pixel, the time required to acquire a full image is longer than 25 seconds. Furthermore, a three-dimensional image consists of a plurality of two-dimensional images. Assuming 100 2D images are required for a 3D image, it takes longer than 2,500 seconds (˜⅔ hour) to acquire the full signals. It is a serious obstacle in practical applications of the FLIM microscopy, especially for continuous observations of live organisms.
A reliable high-speed fluorescence lifetime measurement scheme is desired for the multi-dimensional FLIM microscopy application. In order to fulfill the speed requirement, a single lifetime determination should be completed within less than a few microseconds. The accuracy and precision should be good enough to reliably determine a short lifetime below 1 ns.